Sumo inhibitors

ABSTRACT

A method of identifying a compound that inhibits the sumoylation of Tcf-4 by proteins of the PIAS (protein inhibitor of activated STAT) (for example, PIASy, PIAS1, PIAS3 or PIASxα) is described. Also provided are compounds identified by this method; inhibitors for sumoylation of Tcf-4 by the PFAS family proteins and Tcf-4 desumoylation agents; inhibitors of transcriptional activity of Tcf-4 comprising the above-described inhibitors and/or desumoylation agents; preventives and/or remedies for colon cancer comprising the above-described inhibitors and/or desumoylation agents or the transcriptional activity inhibitors; a method of inhibiting the transcriptional activity of Tcf-4 characterized by inhibiting the sumoylation of Tcf-4 by the PIAS family protein and/or desumoylating sumoylated Tcf-4; and a method of preventing and/or treating colon cancer.

TECHNICAL FIELD

The present invention relates to a compound or inhibitor that inhibitsboth the sumoylation of Tcf-4 by a protein of the PIAS (proteininhibitor of activated STAT) family and the enhanced transcriptionalactivity of Tcf-4 induced thereby, a desumoylation agent for Tcf-4, anda method for identifying the compound. The present invention alsorelates to a preventive and/or remedy for colon cancer and a method forpreventing and/or treating colon cancer that comprises inhibiting thesumoylation of Tcf-4 induced by a PIAS family protein and/ordesumoylating a sumoylated Tcf-4.

BACKGROUND ART

The term “SUMO” is an abbreviation of “small ubiquitin-like modifier”,and refers to a protein that can be used like a ubiquitin for proteinmodification. The conjugation of SUMO with a target protein is termed“sumoylation”. As target proteins of sumoylation, RanGAP1, PML, IKBA, p53 and the like are known.

Sumoylation is a phenomenon that has recently begun to draw attention,and the significance of sumoylation has not yet been elucidated(non-patent document 1). Some enzymes are known to be involved in theprocess of sumoylation. Such enzymes are different from those involvedin the ubiquitination process (non-patent document 2).

There are at least three types of mammalian SUMOs, SUMO-1, SUMO-2, andSUMO-3. SUMO-1 is activated by an E1 enzyme (Aos/Uba2), then transferredto an E2 conjugation enzyme (Ubc9), and finally conjugated to a targetprotein by an E3 ligase (protein inhibitor of activated STAT (hereinafter, abbreviated as “PIAS”) (non-patent documents 3 and 4) or RanBP2(non-patent document 5)). Sumoylation is reversible, and desumoylationtakes place when, e.g., a SUMO-specific protease is employed (non-patentdocument 6). Several SUMO-specific proteases, such as SENP familyproteins, are known in mammals.

PIAS, which is the E3 ligase involved in sumoylation, is known to havefamily proteins. In mammals, four PIAS family proteins have beenidentified, PIASy, PIAS1, PIAS3, and PIASxα.

PIAS family proteins are involved in the intracellular signaltransduction of cytokines and the like. For example, PIASy is atranscription factor of the Tcf family and sumoylates Lef-1 (Lymphoidenhancer factor 1) which works in the Wnt signaling pathway (non-patentdocument 4). Although PIASy suppresses the transcriptional activity ofLef-1, it does not affect its binding activity to DNA or β-catenin. Onthe other hand, Axam, which is identified as an Axin-associated protein,regulates the Wnt signaling pathway negatively by inducing thedegradation of β-catenin (non-patent document 7). Axam is one of theSENP family proteins, and the regulatory functions of Axam in the Wntsignaling pathway require the desumoylation activity thereof (non-patentdocument 8).

PIAS1 and PIAS3 have been identified respectively as auxiliary factorsthat inhibit transcription control factors, STAT1 and STAT3, frombinding to DNA (non-patent document 9). PIAS1 sumoylates the p53tumor-suppressor gene product (non-patent document 3). PIASxα regulatesandrogen receptor (AR)-dependent transcription negatively by binding toAR (non-patent document 10).

As described above, it has been suggested that a target protein issumoylated by the PIAS family proteins, which results in the functionalalteration of the target protein. This has led to the PIAS familyproteins receiving attention as sumoylation enzymes.

References cited herein are described below.

-   Non-patent document 1: Saitoh, T., Experimental Medicine (2001),    vol. 19, p. 112-119.-   Non-patent document 2: Muller, S. et al., Nature Reviews Molecular    Cell Biology (2001) 2: 202-210.-   Non-patent document 3: Kahyo, T. et al., Molecular Cell (2001) 8:    713-718.-   Non-patent document 4: Sachdev, S. et al., Genes and    Development (2001) 15: 3088-3103.-   Non-patent document 5: Kirsh, O. et al., EMBO Journal (2002) 21:    2682-2691.-   Non-patent document 6: Yeh, E. et al., Gene (2000) 248: 1-14.-   Non-patent document 7: Kadoya, T. et al., Molecular and Cellular    Biology (2002) 22: 3803-3819.-   Non-patent document 8: Kadoya, T. et al., Journal of Biological    Chemistry (2000) 275: 37030-37037.-   Non-patent document 9: Liu, B. et al., Proceedings of The National    Academy of Sciences of The United States of America (1998) 95:    10626-10631.-   Non-patent document 10: Kotaja, N. et al., Molecular    Endocrinology (2000) 14: 1986-2000.-   Non-patent document 11: Korinek, B., Science (1997) 275: 1784-1787.-   Non-patent document 12: Morin, P. J. et al., Science (1997) 275:    1787-1790.-   Non-patent document 13: Vogelstein, K. et al., Cell (1996) 87:    159-170.-   Non-patent document 14: Tetsu, O. et al., Nature (1999) 398:    422-426.

DISCLOSURE OF THE INVENTION

In the present invention, concentrated studies have been undertaken withthe following aims: isolating a protein that is sumoylated by a proteinof the PIAS family, regulating the sumoylation thereof, and providingmeans for controlling diseases resulting from the functional alterationof the protein caused by sumoylation. The present invention has beenachieved by finding out that Tcf-4, a transcription factor, issumoylated by PIAS family proteins such as PIASy, PIAS1, PIAS3, andPIASxα, and that the Tcf-4 function is enhanced by sumoylation.

Accordingly, one aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) contacting PIAS and/or Tcf-4 with a testcompound under conditions allowing for the interaction of the testcompound with PIAS and/or Tcf-4, and (ii) determining whether the testcompound inhibits the sumoylation of Tcf-4 by PIAS, by employing asystem that uses a signal and/or marker capable of detecting sumoylationof Tcf-4 to detect the presence, absence, or alteration of the signaland/or marker.

A further aspect of the present invention relates to the aboveidentification method, wherein PIAS is PIASy, PIAS1, PIAS3, or PIASxα.

A still further aspect of the present invention relates to the aboveidentification method, wherein PIAS is PIASy.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) using the cells described in (A), (B), (C),or (D), below, and contacting the cells with a test compound, and (ii)determining whether the test compound inhibits the sumoylation of Tcf-4by PIAS, by detecting the sumoylated Tcf-4 or by detecting the presence,absence, or alteration of a signal and/or marker for detectingsumoylation of Tcf-4, wherein cells (A), (B), (C), and (D) are asfollows:

-   -   (A): cells that have co-expressed PIAS, SUMO, and Tcf-4 (to        which a signal and/or marker for detecting sumoylation of Tcf-4        is introduced),    -   (B): cells that have co-expressed PIAS, Tcf-4, and SUMO (to        which a signal and/or marker for detecting sumoylation of Tcf-4        is introduced),    -   (C): cells that have co-expressed PIAS, Tcf-4 (to which a signal        and/or marker for detecting sumoylation of Tcf-4 is introduced),        and SUMO (to which the signal and/or marker is introduced), and    -   (D): cells that have co-expressed PIAS, SUMO, and Tcf-4.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) using the cells described in (E) or (F),below, and contacting the cells with a test compound, and (ii)determining whether the test compound inhibits the sumoylation of Tcf-4by PIAS by measuring a fluorescent intensity of the green fluorescentprotein (GFP) after adding a luciferase substrate, wherein cells (E) and(F) are as follows:

-   -   (E): cells that have co-expressed a fused protein of Tcf-4 and        GFP, a fused protein of SUMO and luciferase, and PIAS; and    -   (F): cells that have co-expressed a fused protein of Tcf-4 and        luciferase, a fused protein of SUMO and GFP, and PIAS.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) bringing PIAS, E1 SUMO-activating enzyme,E2 SUMO-conjugation enzyme, SUMO or SUMO to which a signal and/or markerfor detecting sumoylation of Tcf-4 is introduced, and a test compound,to contact with Tcf-4 or Tcf-4 immobilized on a solid phase, and (ii)determining whether the test compound inhibits the sumoylation of Tcf-4by PIAS, by detecting the sumoylated Tcf-4, or by detecting thepresence, absence, or alteration of the signal and/or marker.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) using proteins described in (a) or (b),below, and contacting the proteins with a test compound, and (ii)determining whether the test compound inhibits the sumoylation of Tcf-4by PIAS, by detecting the presence, absence, or alteration of a signaland/or marker for detecting sumoylation of Tcf-4, wherein proteins (a)and (b) are as follows:

-   -   (a): PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation        enzyme, SUMO (to which a signal and/or marker for detecting        sumoylation of Tcf-4 is introduced), and Tcf-4 (to which a        signal and/or marker for detecting the signal and/or marker        introduced to the SUMO is introduced); and    -   (b): PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation        enzyme, Tcf-4 (to which a signal and/or marker for detecting        sumoylation of Tcf-4 is introduced), and SUMO (to which a signal        and/or marker for detecting the signal and/or marker introduced        to the Tcf-4 is introduced).

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) bringing PIAS, E1 SUMO-activating enzyme,E2 SUMO-conjugation enzyme, SUMO labeled with a radioactive isotope,biotin, or a peptide-tag, and a test compound to contact with Tcf-4immobilized to a solid phase; and (ii) determining whether the testcompound inhibits the sumoylation of Tcf-4 by PIAS by measuring thesubstance used for labeling.

A still further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) bringing PIAS, E1 SUMO-activating enzyme,E2 SUMO-conjugation enzyme, SUMO (labeled with europium), Tcf-4 (labeledwith a substance radiating secondary fluorescence upon excitation byfluorescence irradiated by europium), and a test compound to reaction;and (ii) determining whether the test compound inhibits the sumoylationof Tcf-4 by PIAS by irradiating light of a wavelength that exciteseuropium and then measuring the secondary fluorescence intensity.

A further aspect of the present invention relates to a method foridentifying a compound that inhibits the sumoylation of Tcf-4 by PIAS,comprising the steps of: (i) bringing PIAS, E1 SUMO-activating enzyme,E2 SUMO-conjugation enzyme, and a test compound to react with one of thefollowing: a fused protein of Tcf-4 and luciferase as well as a fusedprotein of SUMO and green fluorescent protein (GFP), or a fused proteinof Tcf-4 and GFP as well as a fused protein of SUMO and luciferase, and(ii) determining whether the test compound inhibits the sumoylation ofTcf-4 by PIAS by measuring the fluorescence intensity of the GFP afteradding a luciferase substrate.

A further aspect of the present invention relates to a compound that isobtained by any one of the above identification methods.

A still further aspect of the present invention relates to a compoundthat inhibits the sumoylation of Tcf-4 by PIAS.

A further aspect of the present invention relates to a compound thatinhibits the sumoylation of Tcf-4 by PIASy, PIAS1, PIAS3, or PIASxα.

A further aspect of the present invention relates to a compound thatinhibits the sumoylation of Tcf-4 by PIASy.

A still further aspect of the present invention relates to an inhibitorthat inhibits the sumoylation of Tcf-4 by PIAS.

A further aspect of the present invention relates to an inhibitor thatinhibits the sumoylation of Tcf-4 by PIASy, PIAS1, PIAS3, or PIASxα.

A further aspect of the present invention relates to an inhibitor thatinhibits the sumoylation of Tcf-4 by PIASy.

A still further aspect of the present invention relates to adesumoylation agent for Tcf-4.

A further aspect of the present invention relates to an inhibitor oftranscriptional activity of Tcf-4, comprising any one of the abovesumoylation inhibitors and/or the above desumoylation agent.

A further aspect of the present invention relates to a method forinhibiting the transcriptional activity of Tcf-4, comprising inhibitingthe sumoylation of Tcf-4 by PIAS, and/or desumoylating Tcf-4.

A further aspect of the present invention relates to the above methodfor inhibiting the transcriptional activity of Tcf-4, wherein PIAS isPIASy, PIAS1, PIAS3, or PIASxα.

A still further aspect of the present invention relates to the abovemethod for inhibiting the transcriptional activity of Tcf-4, whereinPIAS is PIASy.

A further aspect of the invention relates to a preventive and/or remedyfor colon cancer, comprising any one of the above sumoylation inhibitorsand/or the above desumoylation agent, or the above inhibitor oftranscriptional activity of Tcf-4.

A further aspect of the present invention relates to a method forpreventing and/or treating colon cancer, comprising inhibitingsumoylation of Tcf-4 by PIAS, and/or desumoylating Tcf-4.

A further aspect of the present invention relates to the above methodfor preventing and/or treating colon cancer, wherein PIAS is PIASy,PIAS1, PIAS3, or PIASxα.

A still further aspect of the present invention relates to the abovemethod for preventing and/or treating colon cancer, wherein PIAS isPIASy.

A further aspect of the present invention relates to a medicinalcomposition that comprises at least one member selected from the groupconsisting of: any of the above compounds, any of the above sumoylationinhibitors, the above desumoylation agent, and the above inhibitor oftranscriptional activity of Tcf-4.

A further aspect of the present invention relates to a preventive and/orremedy for colon cancer that comprises at least one member selected fromthe group consisting of: any of the above compounds, any of the abovesumoylation inhibitors, the above desumoylation agent, and the aboveinhibitor of transcriptional activity of Tcf-4.

A still further aspect of the present invention relates to a method forpreventing and/or treating colon cancer, wherein the method uses atleast one member selected from the group consisting of: any of the abovecompounds, any of the above sumoylation inhibitors, the abovedesumoylation agent, and the above inhibitor of transcriptional activityof Tcf-4.

A further aspect of the present invention relates to a reagent kit thatcomprises at least one of the following: PIAS and/or Tcf-4; apolynucleotide encoding PIAS and/or a polynucleotide encoding Tcf-4; ora vector comprising a polynucleotide encoding PIAS and/or a vectorcomprising a polynucleotide encoding Tcf-4.

A further aspect of the present invention relates to the above reagentkit, wherein PIAS is PIASy, PIAS1 , PIAS3, or PIASxα.

A still further aspect of the present invention relates to the abovereagent kit, wherein PIAS is PIASy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B illustrate that the conjugation of SUMO-1 to Tcf-4was accelerated by PIASy. In FIG. 1A, conjugation of SUMO-1 to TCF-4 wascarried out using cells in which their respective genes had beentransfected and was detected by Western Blotting using an anti-Mycantibody. In the figure, “WT” refers to a wild-type PIASy, and “CA”refers to a PIASy wherein cysteines (C) at positions 342 and 347 in theamino acid sequence of PIASy were substituted by alanines (A). In FIG.1B, conjugation of SUMO-1 to TCF-4 was carried out using cells in whichthe respective genes had been transfected. After lysis of the cells,Myc-Tcf-4 was immunoprecipitated with an anti-Myc antibody, and therecovered protein was detected by Western Blotting using an anti-Mycantibody and an anti-HA antibody.

FIG. 2 illustrates that PIASy by itself accelerates the transcriptionalactivity of Tcf-4, and also promotes the β-catenin- or Wnt-3a-dependenttranscriptional activity of Tcf-4. The transcriptional activity of Tcf-4was measured using a firefly luciferase gene as a reporter gene. In thefigure, the longitudinal axis indicates relative luciferase induction,expressed as a fold increase of the luciferase activity. The values inthe figure are the amount of each plasmid added (μg).

FIG. 3 illustrates that SUMO-1 accelerates the β-catenin-dependenttranscriptional activity of Tcf-4. The values in the figure are theamounts of the respective plasmids added (μg).

FIG. 4 illustrates that acceleration of the β-catenin-dependenttranscriptional activity of Tcf-4 is inhibited by Axam, which hasdesumoylation activity, but is not inhibited by an Axam mutant that doesnot have desumoylation activity. In the figure, “WT” refers to wild-typeAxam, and “CS” refers to an Axam mutant in which cysteine (C) atposition 547 in the amino acid sequence was substituted by serine (S),and “72-588”, “72-400”, and “381-588” refer to deletion-type Axammutants.

FIG. 5 is a schematic diagram showing the structures of human PIASy,human Tcf-4, Axam, and various mutants of Axam.

FIG. 6 illustrates that PIASy accelerates the conjugation of SUMO-1 toTcf-4 in vitro, but that PIASyCA does not conjugate SUMO-1 to Tcf-4. Thevalues shown on the left side represent the molecular weights.

FIG. 7 illustrates that any of PIASy, PIAS1, PIAS3, and PIASxαaccelerates the conjugation of SUMO-1 to Tcf-4.

DETAILED DESCRIPTION OF INVENTION

The present invention claims the benefit of priority of Japanese PatentApplication No. 2002-55796 and No. 2002-271527, which are incorporatedherein by reference.

Technical and scientific terms used herein have the meanings asunderstood generally by those skilled in the art, unless otherwisedefined. Reference is made herein to a variety of methods that arewell-known to those skilled in the art. Data from publications and thelike that discloses such cited well-known methods are deemed completelyincorporated herein in their entirety by reference.

A mode of embodiment of the present invention may be described in moredetail hereafter. The following detailed description is illustrative andmerely explanatory, and it dose not limit the scope of the presentinvention.

In the present invention, it has been found that the sumoylation oftranscriptional factor Tcf-4 is accelerated by PIAS family proteins suchas PIASy, PIAS1, PIAS3, or PIASxα, and that the sumoylation enhances theTcf-4 function. Tcf-4 is a DNA-binding protein belonging to the Tcffamily. Tcf-4 is located downstream of the Wnt signaling pathway, andacts as a transcriptional factor by binding to β-catenin.β-catenin-dependent enhancement of the transcriptional activity of Tcf-4was increased synergistically by PIASy. Wnt signal-dependent enhancementof the transcriptional activity of Tcf-4 was also increasedsynergistically by PIASy, where the Wnt signal shows the enhancingeffect on the signal by suppressing the degradation of β-catenin. Inaddition, it has been found that the β-catenin-dependent enhancement ofthe transcriptional activity of Tcf-4 was inhibited by an Axam proteinwith desumoylation activity (non-patent document 7 and non-patentdocument 8), which revealed that the enhancement of the Tcf-4 functionresults from sumoylation. Although these results were obtained by use ofPIASy, it is believed that the transcriptional activity of Tcf-4 can besimilarly enhanced by PIAS1, PIAS3, and PIASxα, since PIAS1, PIAS3, andPIASxα also provide sumoylation to Tcf-4.

Therefore, it is found that an enhanced function of Tcf-4, e.g. itstranscriptional activity, can be downregulated by inhibiting Tcf-4sumoylation and/or by desumoylating sumoylated Tcf-4. A signaltransduction system via β-catenin/Tcf-4 is believed to be involved incell proliferation. Accordingly, suppression of Tcf-4-associated cellproliferation can be achieved by inhibiting Tcf-4 sumoylation and/or bydesumoylation of sumoylated Tcf-4.

Tcf-4 is expressed in normal as well as neoplastic intestine epithelialtissues, and in all colon cancer cell lines examined (non-patentdocument 11). Further, the following have been reported: thattranscriptional activity due to the β-catenin/Tcf complex is constantlyactivated in many cases of colon cancer (non-patent document 11); that aβ-catenin/Tcf signal is activated by mutation of the β-catenin or tumorsuppressor gene APC (Adenomatous poliposis coli)(non-patent document 12and non-patent document 13) in colon cancer; and that in colon cancercells, β-catenin regulates the expression of cyclin D1, which isassociated with cell cycle progression (non-patent document 14).

Since there seems to be a close relationship between enhancement oftranscriptional activity in the β-catenin/Tcf signal transduction systemand colon cancer, it is believed that colon cancer can be treated bymeans of inhibiting the transcriptional activity of Tcf-4 by inhibitingTcf-4 sumoylation and/or by desumoylating sumoylated Tcf-4.

Based on the above findings, according to the present invention thefollowing can be provided: an inhibitor of Tcf-4 sumoylation by PIAS; adesumoylation agent for sumoylated Tcf-4; an inhibitor oftranscriptional activity of Tcf-4 comprising the sumoylation inhibitorand/or the desumoylation agent; a preventive and/or remedy for coloncancer comprising the sumoylation inhibitor and/or the desumoylationagent or the inhibitor of transcriptional activity; a method forinhibiting the transcriptional activity of Tcf-4 comprising inhibitingTcf-4 sumoylation by PIAS and/or desumoylating sumoylated Tcf-4; and amethod for preventing and/or treating colon cancer comprising inhibitingTcf-4 sumoylation by PIAS and/or desumoylating sumoylated Tcf-4.

In the present invention, the term “PIAS” refers to a protein belongingto the PIAS family proteins. In the present invention, the E3 ligasethat associates with Tcf-4 sumoylation may be any PIAS, preferablyPIASy, PIAS1, PIAS3, or PIASxα, and more preferably PIASy. Also, two ormore PIAS proteins may be used as the E3 ligase.

In the present invention, the term “SUMO” includes all members of theSUMO family, such as SUMO-1, SUMO-2, and/or SUMO-3.

Herein, the term “inhibitor of transcriptional activity of Tcf-4” refersto a substance that ultimately inhibits Tcf-4-associated transcriptionalactivity by acting in some manner on a transcriptional activationapparatus including Tcf-4, where the apparatus comprises, e.g., bindingof Tcf-4 to a specific DNA sequence or binding of Tcf-4 to anotherfactor that carries out transcription in association with Tcf-4.

According to the present invention, based on the above information,there is provided a method for identifying a compound that inhibits thesumoylation of Tcf-4 by PIAS. A compound that inhibits the sumoylationof Tcf-4 by PIAS can be identified by selecting conditions allowing forthe interaction of PIAS and/or Tcf-4 with a test compound, contactingPIAS and/or Tcf-4 with the test compound under such conditions, and thendetecting the sumoylation of Tcf-4.

Detection of Tcf-4 sumoylation can be conducted by introducing into theabove identification method a system that uses a signal and/or markercapable of detecting Tcf-4 sumoylation, and then detecting the signaland/or marker. Such signal and/or marker may be used by introducing thesignal and/or marker to SUMO and/or Tcf-4. Regarding SUMO, any SUMO maybe used as long as it belongs to the SUMO family. SUMO-1 is preferablyused. The term “signal” as used herein refers to a substance that isdirectly detectable by its physical or chemical properties, and the term“marker” refers to a substance that is indirectly detectable when itsphysical or biological properties are used as an indicator. Examples ofa signal include luciferase, green fluorescein protein (hereinafter,abbreviated as GFP), europium, and a radioactive isotope (¹²⁵I, ³H, ¹⁴C,³⁵S, etc). Examples of a marker include a reporter gene (such as thechloramphenicol acetyltransferase (CAT) gene), a tag for detection (suchas 6×His-tag, Myc-tag, EE-tag, Express-tag, V5-tag), or a knownsubstance such as biotin. A signal or marker is not limited to the aboveexamples, and any substance may be used as long as it makes it possibleto detect the sumoylation of Tcf-4 by PIAS. Methods for detecting suchsignals or markers are well known to those skilled in the art.

Sumoylated Tcf-4 may be also detected using, e.g., an anti-Tcf-4antibody. When Tcf-4 has been previously modified with another proteinor peptide, sumoylated Tcf-4 can be detected using an antibody for theprotein or peptide in question. Sumoylation of Tcf-4 is detectable,e.g., by detecting the difference in molecular weight between sumoylatedTcf-4 and non-sumoylated Tcf-4 utilizing variations in their respectivemobilities in electrophoresis, using a known protein detection methodsuch as Western Blotting.

Sumoylation of Tcf-4 may also be measured using the transcriptionalactivity of Tcf-4 as an indicator, since the sumoylation of Tcf-4enhances the transcriptional activity thereof. The transcriptionalactivity of Tcf-4 can be measured by a known method for measuring theactivity of a transcriptional factor using a reporter gene, e.g. areporter plasmid having DNA with a Tcf-recognizable nucleotide sequencein its upstream (hereinafter, also referred to as a “Tcf-responsive typeplasmid”).

A compound inhibiting the sumoylation of Tcf-4 can be obtained accordingto the above identification method by comparing an amount of Tcf-4sumoylated under conditions in which a test compound is not present withan amount of Tcf-4 sumoylated under conditions in which the testcompound is present, and selecting the test compound with respect to theamount that decreases. Such comparison is carried out by detecting thepresence, absence, or alteration of a signal and/or marker capable ofdetecting sumoylation of Tcf-4. Alternatively, such a comparison can beconducted by measuring the difference in molecular weight betweensumoylated Tcf-4 and non-sumoylated Tcf-4 using a known method, such asWestern Blotting.

The identification method can be carried out utilizing a well-knownpharmaceutical screening system. PIAS, Tcf-4, and SUMO used in theidentification method may be the following: products in cells that haveexpressed PIAS, Tcf-4, and SUMO by genetic engineering techniques;synthetic products in a cell-free system; chemically synthesizedproducts; products prepared from cells or from any biological samples;or products that are further purified from the foregoing. Further, aslong as sumoylation of Tcf-4 by PIAS is not inhibited, PIAS, Tcf-4, andSUMO may be those to which another protein or peptide (such asglutathione S-transferase (GST), luciferase, GFP, β-galactosidase, Fcfragments of immunoglobulin such as IgG, His-tag, Myc-tag, EE-tag,Express-tag, V5-tag, or Flag-tag) is ligated directly or indirectly viaa linker peptide to the N-terminus side or C-terminus side of PIAS,Tcf-4, or SUMO by using genetic engineering techniques. Examples of testcompounds include compounds derived from chemical libraries or naturalproducts, or compounds obtained by drug designing based on thethree-dimensional structures of PIAS and Tcf-4.

Any of the following systems can be used as the above identificationmethod: a system where PIAS, Tcf-4, and SUMO are expressed in cells bygenetic engineering techniques to induce sumoylation in the cells (insitu system), or a system where PIAS, Tcf-4, and SUMO are employed toinduce sumoylation in vitro. In order to induce sumoylation in vitro,further addition of an E1 SUMO-activating enzyme and/or an E2SUMO-conjugation enzyme other than PIAS, Tcf-4 and SUMO is preferred.

An example of the in situ system includes a system that uses the cellsdescribed in (A), (B), (C), or (D) below, brings the cells to contactwith a compound, and then detects the sumoylated Tcf-4, or detects thepresence, absence, or alteration of a signal and/or marker for detectingTcf-4 sumoylation, wherein cells (A), (B), (C), and (D) are as follows:

-   -   (A): cells that have co-expressed PIAS, SUMO, and Tcf-4 to which        a signal and/or marker for detecting sumoylation of Tcf-4 is        introduced;    -   (B): cells that have co-expressed PIAS, Tcf-4, and SUMO to which        a signal and/or marker for detecting sumoylation of Tcf-4 is        introduced;    -   (C): cells that have co-expressed PIAS, Tcf-4 to which a signal        and/or marker for detecting sumoylation of Tcf-4 is introduced,        and SUMO to which the signal and/or marker is introduced; and    -   (D): cells that have co-expressed PIAS, SUMO, and Tcf-4.

For the above cells, cells that are generally used for gene transfectioncan be preferably used. More preferably used are animal cells, and evenmore preferably used are human colon cancer cells.

As a specific system thereof, a compound that inhibits the sumoylationof Tcf-4 by PIAS can be identified by the following method: providingcells that have expressed PIAS, Myc-tagged Tcf-4, and SUMO; contactingthe cells with a test compound; conducting electrophoresis after acommon pretreatment of the cells; and detecting bands corresponding tothe sumoylated Tcf-4 by Western Blotting using an anti-Myc antibody (seeExample 1).

It is also possible to identify a compound that inhibits the sumoylationof Tcf-4 by PIAS as well as the transcriptional activity of Tcf-4, bythe following method: providing cells that have expressed HA-taggedTcf-4, PIAS, SUMO, and β-catenin, through a Tcf-responsive type plasmid;contacting the cells with a test compound; and detecting a signal and/ormarker derived from the Tcf-response type plasmid (see Example 2).

In the case of a method using Renilla luciferase and GFP, cells thatconstitutively express a fused protein of GFP and Tcf-4 (GFP-Tcf-4) aswell as a fused protein of Renilla luciferase and SUMO (Renilla-SUMO)are employed. When a Renilla luciferase substrate is added to cells in astate where Renilla-SUMO has been conjugated to GFP-Tcf-4 by sumoylationby PIAS, light generated by the luminous reaction of the Renillaluciferase irradiates onto the Tcf-4-linked GFP, whereby the GFP emitssecondary fluorescence. Measurement of the secondary fluorescenceintensity enables the quantification of Tcf-4 sumoylation in the cells.Thus, by contacting the cells with a test compound, and then measuringthe fluorescence intensity of GFP after a specified period of time, itis possible to determine the level of Tcf-4 sumoylation. This method ishighly useful, since it is possible to prevent an increase in theexisting background in a GFP-Tcf-4/Renilla-SUMO system by applying,e.g., an ecdyson-inducing expression system so as to controlRenilla-SUMO expression. This method, using a combination of a fusedprotein of Tcf-4 and Renilla luciferase (Renilla-Tcf-4) as well as afused protein of SUMO and GFP (GFP-SUMO), may be used in a similarmanner to the method described above. The luciferase is not limited toRenilla luciferase, and any known luciferase may be used. In theseidentification methods, the combination of substances to be fused withTcf-4 and SUMO is not limited to GFP and Renilla luciferase, and anycombination, of substances may be used as long as a signal emitted byone of the substances, for example, fluorescence or the like, acts onthe other substance to cause the emission of a signal that is differentfrom the initial signal, such as, for example, fluorescence of adifferent wavelength.

An example of the in vitro system includes a system in which PIAS, E1SUMO-activating enzyme, E2 SUMO-conjugation enzyme, SUMO or SUMO towhich a signal and/or marker for detecting sumoylation of Tcf-4 isintroduced, and a test compound, are contacted with Tcf-4 or Tcf-4immobilized to a solid phase, and sumoylated Tcf-4 is then detected, orthe presence, absence, or alteration of the signal and/or marker isdetected. PIAS, Tcf-4, SUMO, E1 SUMO-activating enzyme, and E2SUMO-conjugation enzyme may be the following: synthetic products in acell-free system; chemically synthesized products; products preparedfrom cells which have expressed those products by genetic engineeringtechniques, products from any biological samples; or products that arefurther purified from the foregoing.

Examples of a signal and/or marker to be introduced to SUMO include aradioactive isotope, biotin, peptide tag, and the like. Radioactiveisotopes that can be used herein include ¹²⁵I, ³H, ¹⁴C, and ³⁵S. Peptidetags that can be used herein include Flag-tag, HA-tag, Myc-tag, EE-tag,Express-tag, and V5-tag. Introduction of these signals and/or markers toSUMO can be performed by a known method.

Detection of the above signal and/or marker is possible by aconventional method. A radioactive isotope, for example, may be detectedby measuring the radioactivity with a commercially available measuringapparatus. Biotin can be detected by, e.g., binding biotin tostreptavidin that has been labeled with alkaline phosphatase (AP) orhorseradish peroxidase (HRP), adding a substrate for the AP or the HRP,and measuring the enzymatic activity. A peptide tag can be detected byusing an antibody to the peptide tag that is labeled with AP or HRP,binding the antibody to the peptide tag, adding a substrate for the APor the HRP, and measuring the enzymatic activity. As the substrate forAP or HRP, a substrate that is generally known to those skilled in theart may be used.

Examples of a solid phase that may be used herein include, but are notlimited to, a commercially available multi-well plate, agarose orSepharose beads, or SPA beads. Tcf-4 can be immobilized onto a solidphase by a conventional method. For example, when using a fused proteinof Tcf-4 and His-tag, a solid phase pre-coated with nickel ion is used,and then Tcf-4 is immobilized on the solid phase. When using a fusedprotein of Tcf-4 and GST, glutathione is used for the coating of thesolid phase. It is also acceptable to use a commercially availableglutathione-coated multi-well plate or beads. In cases where Tcf-4 isused without being fused with a protein or peptide, or whereimmobilization to a solid phase is not performed via a protein orpeptide fused with Tcf-4, Tcf-4 can be immobilized on the solid phase bypre-coating the solid phase with an anti-Tcf-4 antibody, or by usingcommercially available agarose beads or Sepharose beads that arepre-coated with an anti-Tcf-4 antibody.

As specific methods, methods that use SPA beads, glutathione-Sepharosebeads, and a multi-well plate are described below. However, the presentinvention is not limited to these methods.

In a method using SPA beads, human PIAS, Ubc9, Aos1/Uba2, GST-fusedTcf-4, and SUMO are expressed, e.g., by genetic engineering techniques(Escherichia coli or infection of Baculovirus to insect cells),purified, and used. SUMO is labeled with a radioactive isotope, e.g.,¹²⁵I. These purified proteins and a test compound are mixed, to conductsumoylation of Tcf-4. Glutathione-coated SPA beads (Amersham Corp.) areadded to the reaction solution, and the solution is allowed to stand.During this period, the GST-Tcf-4 binds to the glutathione-coated SPAbeads, and the ¹²⁵I-labeled SUMO then binds to the GST-Tcf-4, wherebythe SPA beads are irradiated by radiation emitted by ¹²⁵I, resulting inthe emission of scintillation florescence. By measuring thisfluorescence, the amount of Tcf-4 sumoylation can be quantified. Sincethe inhibition of sumoylation would lower the measured values, thismethod enables the detection of a compound that inhibits sumoylation.

In a method using glutathione-Sepharose beads, human PIAS, Ubc9,Aos1/Uba2, GST-fused Tcf-4, and SUMO are expressed, e.g., by geneticengineering techniques (Escherichia coli or infection of Baculovirus toinsect cells), purified, and used. SUMO is labeled with a radioactiveisotope, e.g., ¹²⁵I. These purified proteins and a test compound aremixed, to conduct sumoylation of Tcf-4. Glutathione-Sepharose beads(Amersham Corp.) are added to the reaction solution. After stirring, thebeads are washed, to collect the beads to which GST-Tcf-4 has bound. Aliquid scintillator is then added to the collected glutathione-Sepharosebeads to measure the amount of ¹²⁵I-SUMO conjugated to Tcf-4. Sinceinhibition of sumoylation by a test compound would lower theradioactivity in comparison with a case in which the test compound isnot used, this method enables the detection of a compound that inhibitssumoylation.

In a method using a multi-well plate, human PIAS, E1 SUMO-activatingenzyme, E2 SUMO-conjugation enzyme, SUMO, and a fused protein of Tcf-4and His-tag (His-Tcf-4) are expressed, e.g., by genetic engineeringtechniques (Escherichia coli or infection of Baculovirus to insectcells), purified, and used. SUMO is labeled with a radioactive isotope,e.g., ¹²⁵I. His-Tcf-4 is immobilized on, e.g., a Ni⁺-coated 96- or384-well plate. A test compound, E1 SUMO-activating enzyme, E2SUMO-conjugation enzyme, ¹²⁵I-SUMO, and PIAS are added to each well, anda sumoylation reaction is performed. After the reaction, the plate iswashed, and the amount of ¹²⁵I-SUMO conjugated to His-Tcf-4 is measured.Since inhibition of sumoylation by using a test compound would lower theradioactivity in comparison with a case in which the test compound isnot used, this method enables the detection of a compound that inhibitssumoylation.

An example of another in vitro system that can be used herein is asystem using the proteins described in (a) or (b) below, wherein theproteins are contacted with a test compound, followed by detection ofthe presence, absence, or alteration of a signal and/or marker fordetecting sumoylation of Tcf-4, wherein proteins (a) and (b) are asfollows:

-   -   (a): PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation        enzyme, SUMO (to which a signal and/or marker for detecting        sumoylation of Tcf-4 is introduced), and Tcf-4 (to which a        signal and/or marker for detecting the signal and/or marker        introduced to SUMO is introduced); and    -   (b): PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation        enzyme, Tcf-4 (to which a signal and/or marker for detecting        sumoylation of Tcf-4 is introduced), and SUMO (to which a signal        and/or marker for detecting the signal and/or marker introduced        to Tcf-4 is introduced).

As a specific method, a method using europium-labeled SUMO(europium-SUMO), and a fused protein of Tcf-4 and XL665 (XL665-Tcf-4) isexemplified. Human PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugationenzyme, XL665-Tcf-4, and SUMO are expressed, e.g., by geneticengineering techniques (Escherichia coli or infection of Baculovirus toinsect cells), purified, and used. SUMO is labeled with europium by aconventional method. A test compound, XL665-Tcf, E1 SUMO-activatingenzyme, E2 SUMO-conjugation enzyme, europium-SUMO and PIAS are added toeach well, and a sumoylation reaction is carried out. After thereaction, light of 337 nm is irradiated into the wells so as to inducefluorescence emission from europium, and then a secondary fluorescence(665 nm), which is emitted from XL665 as a result of the exposure to theeuropium fluorescence, is measured and the amount of europium-SUMOconjugated to XL665-Tcf-4 is determined. Since inhibition of sumoylationby the test compound would lower the secondary fluorescence intensity incomparison with a case in which the test compound is not used, thismethod enables the detection of a compound that inhibits sumoylation.

As another specific method, a method using a fused protein of Tcf-4 andRenilla luciferase (Renilla-Tcf-4) as well as a fused protein of SUMOand GFP (GFP-SUMO) is exemplified. Human PIAS, E1 SUMO-activatingenzyme, E2 SUMO-conjugation enzyme, Renilla-Tcf-4, and GFP-SUMO areexpressed, e.g., by genetic engineering techniques (Escherichia coli orinfection of Baculovirus to insect cells), purified, and used. A testcompound, Renilla-Tcf-4, E1 SUMO-activating enzyme, E2 SUMO-conjugationenzyme, GFP-SUMO, and PIAS are added to each well of a plate, and asumoylation reaction is carried out. After the reaction, a luciferasesubstrate is added to the well to induce a luminous reaction, and thefluorescence from the excited GFP that results from the luminousreaction is measured, to allow detection of the amount of SUMOconjugated to Tcf-4. Since inhibition of sumoylation by the testcompound would lower the luminescent intensity in comparison with a casein which the test compound is not used, this method enables thedetection of a compound that inhibits sumoylation.

A method using a combination of a fused protein of Tcf-4 and GFP(GFP-Tcf-4) as well as a fused protein of SUMO and Renilla luciferase(Renilla-SUMO) may be also applied similarly to the above method.

Each of the specific examples of identification methods described aboveillustrates an aspect of the present identification method, and thepresent identification method is not limited to these examples. Anymethod that can detect the conjugation of SUMO to Tcf-4 resulting froman interaction of PIAS with Tcf-4 is included in the scope of thepresent invention.

A compound obtained by the above method can be used as an inhibitor ofsumoylation of Tcf-4 by PIAS, and an inhibitor of transcriptionalactivity of Tcf-4 by inhibiting the sumoylation of Tcf-4 by PIAS.Examples of such a compound include a compound that inhibits theinteraction of PIAS with Tcf-4, and a compound that inhibits theactivity of PIAS for Tcf-4 sumoylation. For example, a peptide oroligopeptide comprising an amino acid sequence of a region in which thetwo proteins interact with each other may be exemplified. The peptide oroligopeptide can be identified by designing the peptide or oligopeptideon the basis of the amino acid sequence of PIAS or Tcf-4, synthesizingthe peptide or oligopeptide by a known peptide synthesizing method, andtesting the peptide or oligopeptide according to the above method todetermine whether the thus-obtained peptide or oligopeptide inhibits thebinding of PIAS and Tcf-4 as well as the sumoylation of Tcf-4 by PIAS.Further, an antibody that inhibits the interaction of PIAS with Tcf-4may be exemplified as one of the above compounds. The antibody can beobtained, for example, using a peptide or oligopeptide (comprising anamino acid sequence of a region in which the two proteins interact witheach other) as an antigen, by using antibody preparation methodswell-known in the art.

A compound inhibiting the expression and/or function of PIAS is alsoincluded in the scope of the present invention, since the sumoylation ofTcf-4 by PIAS may be inhibited as a result thereof. Identification of acompound that inhibits PIAS expression can be performed using awell-known screening system for inhibitors of protein expression. As anexample of a compound that inhibits the expression of PIAS, an antisenseoligonucleotide of the PIAS gene may be exemplified. The antisenseoligonucleotide can be obtained by selecting an oligonucleotide thatspecifically inhibits the expression of the PIAS gene, fromoligonucleotides designed on the basis of the nucleotide sequence of thePIAS gene, using an expression system of the PIAS gene. Identificationof a compound that inhibits PIAS function may be performed by the aboveidentification method using PIAS, by selecting a substance that inhibitsa function thereof, such as sumoylation activity.

It has been clarified in the present invention that sumoylated Tcf-4 canhave an enhanced transcriptional activity, and therefore it is furtherexpected that a desumoylation agent, which releases SUMO from sumoylatedTcf-4, is useful for suppression of the transcriptional activity ofTcf-4 as well as for prevention and/or treatment of a disease thatdepends on enhanced transcriptional activity of Tcf-4, such as coloncancer. Examples of a desumoylation agent include Axam, which hasdesumoylation activity and which will be described hereafter in theExamples, or a deletion mutant thereof that retains the same activity.

The thus selected compound, the inhibitor of Tcf-4 sumoylation by PIAS,the desumoylation agent for sumoylated Tcf-4, and the inhibitor oftranscriptional activity of Tcf-4 comprising the inhibitor and/or thedesumoylation agent, may be used as a reagent by using it alone or incombination. Such a reagent is useful for research concerning, e.g.,sumoylation or a β-catenin/Tcf-4 signal transduction system.

The compounds selected above, the inhibitor of Tcf-4 sumoylation byPIAS, the desumoylation agent of sumoylated Tcf-4, and the inhibitor oftranscriptional activity comprising the inhibitor and/or thedesumoylation agent, can be formulated as a medicinal composition uponselection after consideration of the balance between biologicalusefulness and toxicity. Such compounds, an inhibitor of Tcf-4sumoylation, a desumoylation agent for Tcf-4, and an inhibitor oftranscriptional activity of Tcf-4, may be used alone or in combination.

The above compounds, the inhibitor of Tcf-4 sumoylation, thedesumoylation agent for Tcf-4, the inhibitor of transcriptional activityof Tcf-4, and the medicinal composition can be applied to preventionand/or treatment of a cell proliferation-induced disease such as cancer.Furthermore, since it has been reported that Tcf-4 is activated in coloncancer, the above medicinal composition is useful for prevention and/ortreatment of colon cancer.

In terms of the formulation, the inhibitor of Tcf-4 sumoylation by PIAS,the desumoylation agent of sumoylated Tcf-4, the inhibitor oftranscriptional activity comprising the inhibitor and/or thedesumoylation agent, and the medicinal composition, are preferablyformulated in combination with suitable pharmaceutical carriers. Such aformulation comprises a therapeutically effective amount of the abovecompounds, the above inhibitor of sumoylation, the above desumoylationagent, the above inhibitor of transcriptional activity, and/or the abovemedicinal composition, as well as a pharmaceutically acceptable carrieror vehicle. Examples of a carrier include physiological saline, bufferedphysiological saline, dextrose, water, glycerol, ethanol, and a mixturethereof. However, it is not limited thereto. The formulation may beselected according to an administration route, and such formulation iswell-known to those skilled in the art.

The above inhibitor of sumoylation, the above inhibitor oftranscriptional activity, the above desumoylation agent, and the abovemedicinal composition can be used alone or in combination with othercompounds that are therapeutically effective.

In terms of the mode of administration, it may be either systemicadministration or local administration. One preferred mode of systemicadministration is injection, e.g., an intravenous injection. Otherinjection routes, such as subcutaneous, intramuscular, orintraperitoneal injection, may also be used. Another mode ofadministration may be oral administration, so long as an entericformulation or a capsule formulation can be suitably formulated. Inaddition, transmucosal administration or percutaneous administration,which comprises using a penetrant such as bile salt, fusidic acid, orother surfactants, may also be used. In a local administration, formssuch as salve, paste, or gel, may be used.

Suitable dosage ranges can be determined according to the following: theeffectiveness of the above inhibitor of sumoylation, the abovedesumoylation agent, the above inhibitor of transcriptional activity,and the above medicinal composition; the administration route thereof;the characteristics of the formulation; the symptomatic conditions of asubject; and the judgment of a doctor in attendance. Specifically, asuitable dosage may fall, e.g., within a range of 0.1 to 100 μg per 1 kgof the body weight of a subject. However, the dosage may be altered bymeans of common conventional experiments for optimization of a dosagethat are well-known in the art.

Preparation of a pharmaceutical may be carried out by well-knownformulating procedures in accordance with the physical properties of thesubstance employed, for example, peptides, proteins, oligonucleotides,antibodies, or compounds. Specifically, formulations such as powdereddrugs, pills, tablets, capsules, aqueous solutions, liposomalformulations, fat emulsions, clathrates (such as those of cyclodextrin),and the like can be used.

Powdered drugs, pills, capsules, or tablets can be prepared using, forexample, an excipient such as lactose, glucose, sucrose, or mannitol; adisintegrant such as starch or sodium arginate; a lubricant such asmagnesium stearate or talc; a binder such as polyvinylalcohol,hydroxypropyl cellulose, or gelatin; a surfactant such as fatty acidester; and a plasticizer such as glycerin, or the like. For preparationof a tablet or capsule, a solid pharmaceutical carrier is used.

A suspension can be prepared using water; saccharides such as sucrose,sorbitol, or fructose; glycols such as PEG; and oils.

Injectable solutions can be prepared using a carrier comprising a salinesolution, a glucose solution, or a mixture of the salt water and glucosesolution.

Inclusion into a liposome formulation may be conducted in the followingmanner: by dissolving the substance of interest in a solvent (e.g.,ethanol) to make a solution, adding a solution of phospholipidsdissolved in an organic solvent (e.g., chloroform), removing the solventby evaporation and adding a phosphate buffer thereto, agitating thesolution and then subjecting it to sonication followed by centrifugationto obtain supernatant, and finally, filtrating the supernatant forrecovering a liposome.

Fat emulsion can be prepared in the following manner: by mixing thesubstance of interest, an oil ingredient (vegetable oil such as soybeanoil, sesame oil, olive oil, or MCT), an emulsifier (such asphospholipid), and the like; heating the mixture to make a solution;adding water of a required quantity; and then emulsifying orhomogenizing by use of an emulsifier (homogenizer, e.g., a high pressurejet type, an ultrasonic type, or the like). The fat emulsion may be alsolyophilized. For conducting lipid-emulsification, an auxiliaryemulsifier may be added, and examples thereof include glycerin orsaccharides (e.g., glucose, sorbitol, fructose, etc.).

Inclusion into a cyclodextrin formulation may be carried out in thefollowing manner: by dissolving the substance of interest in a solvent(e.g., ethanol); adding a solution of cyclodextrin dissolved in waterunder heating thereto; chilling the solution and filtering theprecipitates; and drying under sterilization. At this time, thecyclodextrin to be used may be appropriately selected from among thosehaving different void sizes (α, β, or γ type) in accordance with thebulkiness of the substance.

The present invention also provides a reagent kit, comprising at leastthe following: PIAS and/or Tcf-4; a polynucleotide encoding PIAS and/ora polynucleotide encoding Tcf-4; or a vector comprising a polynucleotideencoding PIAS and/or a vector comprising a polynucleotide encodingTcf-4. The reagent kit can be used in the above identification methods.PIAS and Tcf-4 may be any of the following: products in cells that haveexpressed PIAS or Tcf-4 by genetic engineering techniques; synthesizedproducts in a cell-free system; chemically synthesized products;products prepared from the cells or any biological samples; or productsthat are further purified from the foregoing. Further, as long assumoylation of Tcf-4 by PIAS is not inhibited, Tcf-4 and PIAS may bethose to which another protein or peptide (such as glutathioneS-transferase (GST), luciferase, green fluorescent protein (GFP),β-galactosidase, immunoglobulin Fc fragments like IgG, His-tag, Myc-tag,or Flag-tag) is ligated directly or indirectly via, e.g., a linkerpeptide to the N-terminus side or C-terminus side of PIAS or Tcf-4 by,e.g., genetic engineering techniques. A polynucleotide encoding PIAS orTcf-4 may be prepared from a human cDNA library by known geneticengineering techniques. A vector comprising a polynucleotide thatencodes PIAS or Tcf-4 is obtained by inserting the above polynucleotideinto a suitable expression vector DNA (such as a vector derived from abacterial plasmid) by known genetic engineering techniques. The reagentkit may further comprise materials necessary for the aboveidentification methods; a signal and/or marker for detecting theinteraction of PIAS with Tcf-4, such as Tcf-4 sumoylation by PIAS; asignal and/or marker for detecting the enhanced function of Tcf-4 byPIAS; SUMO, or another protein- or peptide-modified SUMO; apolynucleotide encoding SUMO; a vector carrying the polynucleotide; or abuffer and the like. In terms of preparation thereof, it is sufficientto use a well-known means for the preparation suitable for eachsubstance to be used.

EXAMPLES

The present invention may be described more particularly in thefollowing examples, but the present invention is not limited to theseexamples.

Plasmids used in the following examples were constructed first.

A cDNA coding for human Tcf-4 having a Myc-tag was inserted into aprotein expression vector for mammalian cells, to construct a Myc-Tcf-4expression plasmid.

A cDNA coding SUMO was inserted into a protein expression vector with anHA-tag sequence for mammalian cells, to construct an HA-SUMO expressionplasmid.

A cDNA coding PIASy having a Flag-tag was inserted into a proteinexpression vector with a cytomegalovirus promoter for mammalian cells,to construct a Flag-PIASy expression plasmid. A plasmid containingmutated cDNA for substitution of cysteines (C) at positions 342 and 347in the amino acid sequence of PIASy with alanines (A) (PIASyCA) was alsoconstructed.

A cDNA coding for β-catenin (whose GSK-3β phosphorylation site at theN-terminus was substituted with alanine, and which had an HA-tag) wasinserted into a vector pUC19-EF1α at the KpnI/SalI site thereof, toconstruct an HA-β-catenineSA expression plasmid.

cDNAs (FIG. 5) coding for Axam (non-patent document 8) or the deletionmutants thereof were inserted into protein expression vectors formammalian cells, to construct their respective expression plasmids.

Example 1

(Detection of SUMO-1 Conjugation to Tcf-4)

Human embryonic kidney 293 cells (hereinafter, abbreviated as HEK293cell) were seeded in the amount of 3×10⁵ cells in a 35 mm-diameter dish,and incubated for 24 h. After incubation, the cells were transfectedwith 1 μg of the Myc-Tcf-4 expression plasmid, 0.25 μg of the HA-SUMO-1expression plasmid, and 0.02 μg of the Flag-PIASy expression plasmidaccording to the combinations shown in FIGS. 1A and 1B, usingLipofectAMINE 2000 manufactured by GIBCO BRL Corp. Gene transfection wassimilarly performed using the Flag-PIASyCA expression plasmid in placeof the Flag-PIASy expression plasmid. Transfection was performedaccording to the manufacturer's instructions. After the genetransfection, cells were incubated for 40 h, followed by treatment with10% trichloroacetic acid (TCA) containing 2 mM dithiothreitol (DTT), togive a sample for electrophoresis. Tcf-4 with Myc-tag that was expressedin the cells was detected by Western Blotting using an anti-Myc antibody(FIG. 1A).

The HEK 293 cells, which were transfected with each gene by the samemanner as described above to express proteins, were lysed by adding RIPAbuffer (10 mM Na-phosphate buffer, pH 7.2, 150 mM NaCl, 1%Na-deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM DTT, 1 mMphenylmethylsulfonyl fluoride(PMSF), 1 μg/ml aprotinin and leupeptin, 1mM NaF, 0.4 mM Na-orthovanadate, and 10 mM N-ethylmaleimide) to make acell lysate. To the lysate containing a total protein content of 200 μg,was added an anti-Myc antibody, which had been used in the WesternBlotting procedures, and the reaction was carried out for 2 h. Afterthat, ProteinA beads (Amersham Pharmacia Biotech Corp.) were added toimmunoprecipitate the anti-Myc antibody-bound Myc-Tcf-4 protein. Then,the recovered protein was detected by Western Blotting using an anti-Mycantibody and an anti-HA antibody (FIG. 1B).

As a result, in the presence of Tcf-4, SUMO-1, and PIASy in HEK 293cells, a shift in mobility due to conjugation of SUMO-1 to Tcf-4 wasdetected (FIG. 1A). It is believed that the shift in mobility resultsfrom the sumoylation of Tcf-4, because the change was not observed forinactive-form PIASyCA without SUMO activity, and conjugation of SUMO-1with HA-tag was confirmed in the shifted band (FIG. 1B). Accordingly, itwas revealed that sumoylation of Tcf-4 is accelerated by PIASy.

Example 2

(Detection of PIASy Effects on Transcriptional Activity of Tcf-4)

HEK 293 cells were seeded in the amount of 3×10⁵ cells in a 35mm-diameter dish and incubated for 24 h. After incubation, the cellswere transfected with a Tcf-responsive type plasmid (0.5 μg) insertedusing LipofectAMINE 2000. At this time, a Wnt-3a expression plasmid (0.5μg), a PIASy or PIASyCA expression plasmid, and an HA-β-catenineSAexpression plasmid, were simultaneously added to some of the cells,according to the combinations and quantities (μg) shown in FIG. 2. Afterthe transfection with plasmids, incubation was performed for 40 h, andthen the activity of firefly luciferase derived from the Tcf-responsivetype plasmid, which was generated in the cells, was detected by alumiphotometer manufactured by Futaba Medical Corp. (Tokyo), whereinPicaGene manufactured by Toyo Beanet Co. was used as a substrate. Anerror ascribable to a difference in transfection effectiveness wascorrected by measuring the activity of pME18S/lacZ-derivedβ-galactosidase (FIG. 2).

In the HEK 293 cells, the reporter activity was enhanced by expressionof β-catenin or Wnt-3. PIASy solely enhanced the reporter activity, andalso synergistically increased the β-catenin- or Wnt-3-dependentreporter activity. On the other hand, inactive-form PIASyCA withoutsumoylation activity suppressed an increase of β-catenin-dependentreporter activity. Thus, it was revealed that PIASy solely enhances thetranscriptional activity of Tcf-4 and also that PIASy synergisticallyincreases the β-catenin- or Wnt-3-dependent transcriptional activity ofTcf-4.

Example 3

(Effects of PIASy, SUMO-1, or Axam on β-catenin-dependentTranscriptional Activity of Tcf-4)

HEK293 cells were seeded in the amount of 3×10⁵ cells in a 35mm-diameter dish and incubated for 24 h. After incubation, the cellswere transfected with a Tcf-responsive type plasmid (0.5 μg), apME18S/lacZ (0.5 μg), a Myc-Tcf-4 expression plasmid (0.1 μg), and anHA-β-cateninSA expression plasmid (30 ng) using LipofectAMINE 2000. Atthat time, PIASy, SUMO-1, and Axam protein (in some cases, a mutantthereof) with desumoylation activity were simultaneously added in theamount of 0.5 μg. After incubation for 40 h, the activity of fireflyluciferase derived from the Tcf-responsive type plasmid, which wasgenerated in the cells, was detected by a lumiphotometer manufactured byFutaba Medical Corp. (Tokyo), wherein PicaGene manufactured by ToyoBeanet Co. was used as a substrate. Also, an error ascribable to thedifference in transfection effectiveness was corrected by measuring theactivity of pME18S/lacZ-derived β-galactosidase (FIGS. 3 to 5).

SUMO-1 synergistically accelerated the β-catenin-dependenttranscriptional activity of Tcf-4 (FIG. 3). Accordingly, it isconsidered that sumoylation is involved in the enhancement oftranscriptional activity of Tcf-4.

A full-length Axam protein with desumoylation activity showedsuppression of the β-catenin-dependent transcriptional activity of Tcf-4(FIG. 4 and FIG. 5). In contrast, a mutant in which cysteine (C) atposition 547 in the amino acid sequence of Axam had been substitutedwith serine (S) (a mutant exhibiting no desumoylation activity), did notsuppress the β-catenin-dependent transcriptional activity of Tcf-4.Among the mutants of Axam in which amino acid residues were deleted atthe N-terminal side, Axam (72-400) increased the β-catenin-dependenttranscriptional activity of Tcf-4, while Axam (72-588) and Axam(381-588) showed suppression of the activity although their levels wereweaker than that of the full-length Axam.

From the above results, it was revealed that the acceleration ofsumoylation induces enhancement of the transcriptional activity ofTcf-4, and in contrast, the acceleration of desumoylation inducessuppression of the transcriptional activity of Tcf-4. Therefore, thetranscriptional activity of Tcf-4 can be suppressed by inhibiting Tcf-4sumoylation, which enables the prevention and/or treatment of coloncancer, since it is reported that Tcf-4 is activated in colon cancer.

Example 4

(Detection of SUMO-1 Conjugation to Tcf-4 in vitro)

cDNA coding for human Tcf-4 with GST-tag was inserted into a Baculovirusvector to construct a vector, and then GST-Tcf-4 was produced using Sf9cells. GST-Aos1/His6-Uba2 (E1) was similarly produced using Sf-9 cells.SUMO-IGG (G denotes glycine), His6-Ubc9 (E2), MBP-PIASy (WT), and PIASy(PIASyCA: C342, 347A)(E3) were produced by Escherichia coli, after eachcDNA had been inserted into a protein expression vector, respectively.

0.3 μg of GST-Tcf-4, 0.5 μg of GST-Aos1/His6-Uba2 (E1), 0.5 μg ofHis6-Ubc9 (E2), and 10 μg of GST-SUMO-IGG were mixed in a sumoylationbuffer solution (50 mM of Tris-HCl buffer, pH 7.5, 1 mM of MgCl₂, 2 mMof DTT, 5 mM of adenosine triphosphate (ATP)) to react at 30° C. for 3h. After that, the Tcf-4 protein was detected by Western Blotting usingan anti-Tcf-4 antibody. However, a shift in mobility ascribable to theconjugation of SUMO-1 to Tcf-4 was not detected. When 0.1 μg or 0.2 μgof MBP-PIASy was further added thereto and the Tcf-4 protein wasdetected similarly, a shift in the mobility was observed. However, ashift in mobility was not detected when MBP-PIASyCA that does not havebinding ability to Ubc9 (FIG. 6) was added. These findings proved thatTcf-4 is sumoylated by PIASy in vitro.

Example 5

(Detection of SUMO-1 Conjugation to Tcf-4 by PIAS)

A human Tcf-4 expression plasmid, a Flag-PIAS3 expression plasmid, anHA-PIAS1 expression plasmid, and an HA-PIASxα expression plasmid wereconstructed using a recombinant DNA technique. Similarly to Example 1,these plasmids, together with an HA-SUMO-1 expression plasmid, weretransfected in HEK 293 cells. After incubation, the cells were treatedwith 10% TCA to give a precipitate. The precipitate was subjected toelectrophoresis to detect the sumoylation of Tcf-4 using an anti-Tcf-4antibody, anti-Flag antibody, and anti-HA antibody (FIG. 7).

As a result, in the presence of Tcf-4 and SUMO-1 in the HEK 293 cells, ashift in mobility due to PIASy, PIAS1, PIAS3, and PIASxα was detected(FIG. 7, lanes 4 to 7). This shift in mobility results from theconjugation of SUMO-1 to Tcf-4. It has thus been proved that thesumoylation of Tcf-4 is accelerated by not only PIASy, but also byPIAS1, PIAS3, and PIASxα.

INDUSTRIAL APPLICABILITY

In the present invention, it has been found that transcriptional factorTcf-4 is sumoylated by PIAS, such as PIASy, PIAS1, PIAS3, or PIASxα, andthat the sumoylation enhances the Tcf-4 function. It is known that Tcf-4is a transcriptional factor that is located downstream of a β-cateninsignal, and that it is activated in colon cancer. Thus, inhibiting thesumoylation of Tcf-4 by PIAS or desumoylating sumoylated Tcf-4 cansuppress the enhanced transcriptional activity of Tcf-4, which may allowthe prevention and/or treatment of colon cancer. The present inventionis therefore extremely useful for preventing and/or treating coloncancer, and for research concerning sumoylation or a β-catenin/Tcfsignal transduction system.

1. A method for identifying a compound that inhibits sumoylation ofTcf-4 by PIAS (protein inhibitor of activated STAT), comprising thesteps of: (i) contacting PIAS and/or Tcf-4 with a test compound underconditions allowing for the interaction of the test compound with PIASand/or Tcf-4; and (ii) determining whether the test compound inhibitsthe sumoylation of Tcf-4 by PIAS, by employing a system that uses asignal and/or marker capable of detecting sumoylation of Tcf-4 to detectthe presence, absence, or alteration of the signal and/or marker.
 2. Themethod for identifying a compound according to claim 1, wherein the PIAS(protein inhibitor of activated STAT) is PIASy, PIAS1, PIAS3, or PIASxα.3. The method for identifying a compound according to claim 1, whereinthe PIAS (protein inhibitor of activated STAT) is PIASy.
 4. A method foridentifying a compound that inhibits sumoylation of Tcf-4 by PIAS(protein inhibitor of activated STAT), comprising the steps of: (i)contacting the cells described in (A), (B), (C), or (D) below with atest compound; and (ii) determining whether the test compound inhibitsthe sumoylation of Tcf-4 by PIAS, by detecting sumoylated Tcf-4 or bydetecting the presence, absence or alteration of a signal and/or markerfor detecting sumoylation of Tcf-4; wherein cells (A), (B), (C), and (D)are as follows: (A) cells that have coexpressed PIAS, smallubiquitin-like modifier (SUMO), and Tcf-4 to which a signal and/ormarker for detecting sumoylation of Tcf-4 is introduced; (B) cells thathave coexpressed PIAS, Tcf-4, and SUMO to which a signal and/or markerfor detecting sumoylation of Tcf-4 is introduced; (C) cells that havecoexpressed PIAS, Tcf-4 to which a signal and/or marker for detectingsumoylation of Tcf-4 is introduced, and SUMO to which the signal and/ormarker is introduced; and (D) cells that have coexpressed PIAS, SUMO,and Tcf-4.
 5. A method for identifying a compound that inhibitssumoylation of Tcf-4 by PIAS (protein inhibitor of activated STAT),comprising the steps of: (i) contacting the cells described in (E) or(F) below with a test compound; and (ii) determining whether the testcompound inhibits the sumoylation of Tcf-4 by PIAS, by measuring afluorescence intensity of a green fluorescent protein (GFP) after addinga luciferase substrate; wherein cells (E) and (F) are as follows: (E)cells that have coexpressed a fused protein of Tcf-4 and GFP, a fusedprotein of small ubiquitin-like modifier (SUMO) and luciferase, andPIAS; and (F) cells that have co-expressed a fused protein of Tcf-4 andluciferase, a fused protein of SUMO and GFP, and PIAS.
 6. A method foridentifying a compound that inhibits sumoylation of Tcf-4 by PIAS(protein inhibitor of activated STAT), comprising the steps of:(i)bringing PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation enzyme,small ubiquitin-like modifier (SUMO) or SUMO to which a signal and/ormarker for detecting sumoylation of Tcf-4 is introduced, and a testcompound, to contact with Tcf-4 or Tcf-4 immobilized on a solid phase;and (ii) determining whether the test compound inhibits the sumoylationof Tcf-4 by PIAS, by detecting sumoylated Tcf-4 or by detecting thepresence, absence, or alteration of the signal and/or marker.
 7. Amethod for identifying a compound that inhibits sumoylation of Tcf-4 byPIAS (protein inhibitor of activated STAT), comprising the steps of: (i)utilizing proteins described in (a) or (b) below and contacting theproteins with a test compound; and (ii) determining whether the testcompound inhibits the sumoylation of Tcf-4, by detecting the presence,absence, or alteration of a signal and/or marker for detectingsumoylation of Tcf-4; wherein proteins (a) and (b) are as follows: (a)PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation enzyme, smallubiquitin-like modifier (SUMO) to which a signal and/or marker fordetecting sumoylation of Tcf-4 is introduced, and Tcf-4 to which asignal and/or marker for detecting the signal and/or marker introducedto the SUMO is introduced; and (b) PIAS, E1 SUMO-activating enzyme, E2SUMO-conjugation enzyme, Tcf-4 to which a signal and/or marker fordetecting sumoylation of Tcf-4 is introduced, and SUMO to which a signaland/or marker for detecting the signal and/or marker introduced to theTcf-4 is introduced.
 8. A method for identifying a compound thatinhibits sumoylation of Tcf-4 by PIAS (protein inhibitor of activatedSTAT), comprising the steps of: (i) bringing PIAS, E1 SUMO-activatingenzyme, E2 SUMO-conjugation enzyme, small ubiquitin-like modifierlabeled with a radioactive isotope, biotin or a peptide-tag, and a testcompound to contact with Tcf-4 immobilized to a solid phase; and (ii)determining whether the test compound inhibits the sumoylation of Tcf-4by PIAS by measuring the substance used for labeling.
 9. A method foridentifying a compound that inhibits sumoylation of Tcf-4 by PIAS(protein inhibitor of activated STAT), comprising the steps of: (i)bringing PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation enzyme,small ubiquitin-like modifier labeled with europium, Tcf-4 labeled witha substance that radiates secondary fluorescence upon excitation byfluorescence irradiated by europium, and a test compound to reaction;and (ii) determining whether the test compound inhibits the sumoylationof Tcf-4 by PIAS, by irradiating light of a wavelength that exciteseuropium and then measuring the secondary fluorescence intensity.
 10. Amethod for identifying a compound that inhibits sumoylation of Tcf-4 byPIAS (protein inhibitor of activated STAT), comprising the steps of: (i)bringing PIAS, E1 SUMO-activating enzyme, E2 SUMO-conjugation enzyme,and a test compound to react with a fused protein of Tcf-4 andluciferase as well as a fused protein of small ubiquitin-like modifier(SUMO) and a green fluorescent protein (GFP), or with a fused protein ofTcf-4 and GFP as well as a fused protein of SUMO and luciferase; and(ii) determining whether the test compound inhibits the sumoylation ofTcf-4 by PIAS, by measuring the fluorescence intensity of the GFP afteradding a luciferase substrate.
 11. A compound obtained by the method foridentifying a compound according to claim
 1. 12. A compound thatinhibits sumoylation of Tcf-4 by PIAS (protein inhibitor of activatedSTAT).
 13. A compound that inhibits sumoylation of Tcf-4 by PIASy(protein inhibitor of activated STATy), PIAS1, PIAS3, or PIASxα.
 14. Acompound that inhibits sumoylation of Tcf-4 by PIASy (protein inhibitorof activated STATy).
 15. An inhibitor of sumoylation of Tcf-4 by PIAS(protein inhibitor of activated STAT).
 16. An inhibitor of sumoylationof Tcf-4 by PIASy (protein inhibitor of activated STATy), PIAS1, PIAS3,or PIASxα.
 17. An inhibitor of sumoylation of Tcf-4 by PIASy (proteininhibitor of activated STATy).
 18. A desumoylation agent for Tcf-4. 19.An inhibitor, of transcriptional activity of Tcf-4, comprising thesumoylation inhibitor according to claim 15 and/or said desumoylationagent.
 20. A method for inhibiting transcriptional activity of Tcf-4,comprising inhibiting sumoylation of Tcf-4 by PIAS (protein inhibitor ofactivated STAT) and/or desumoylating Tcf-4.
 21. The method forinhibiting transcriptional activity of Tcf-4 according to claim 20,wherein the PIAS (protein inhibitor of activated STAT) is PIASy, PIAS1,PIAS3, or PIASxα.
 22. The method for inhibiting transcriptional activityof Tcf-4 according to claim 20, wherein the PIAS (protein inhibitor ofactivated STAT) is PIASy.
 23. (canceled)
 24. A method for preventingand/or treating colon cancer, comprising inhibiting sumoylation of Tcf-4by PIAS (protein inhibitor of activated STAT) and/or desumoylatingTcf-4.
 25. The method for preventing and/or treating colon canceraccording to claim 24, wherein the PIAS (protein inhibitor of activatedSTAT) is PIASy, PIAS1, PIAS3, or PIASxα.
 26. The method for preventingand/or treating colon cancer according to claim 24, wherein the PIAS(protein inhibitor of activated STAT) is PIASy.
 27. A medicinalcomposition, comprising at least one member selected from the groupconsisting of: the compound according to claim 11, an inhibitor ofsumoylation of Tcf-4 by PIAS (protein inhibitor of activated STAT),PIAS1, PIAS3, PIASxα, or PIASy, a desumoylation agent for Tcf-4, or aninhibitor of transcriptional activity of Tcf-4 which comprises saidinhibitor of sumoylation or said desumoylation agent.
 28. A preventiveand/or remedy for colon cancer, comprising at least one member selectedfrom the group consisting of: the compound according to claim 11, aninhibitor of sumoylation of Tcf-4 by PIAS (protein inhibitor ofactivated STAT), PIAS1 PIAS3, PIASxα, or PIASy, a desumoylation agentfor TcF-4 or an inhibitor of transcriptional activity of Tcf-4 whichcomprises said inhibitor of sumoylation or said desumoylation agent. 29.A method for preventing and/or treating colon cancer, wherein the methoduses at least one member selected from the group consisting of: thecompound according to claim 11, an inhibitor of sumoylation of Tcf-4 byPIAS (protein inhibitor of activated STAT), PIAS1, PIAS3, PIASxα, orPIASy, a desumoylation agent for Tcf-4, or an inhibitor oftranscriptional activity of Tcf-4 which comprises said inhibitor ofsumoylation or said desumoylation agent.
 30. A reagent kit, comprisingat least one of the following: PIAS (protein inhibitor of activatedSTAT) and/or Tcf-4; or a polynucleotide encoding PIAS and/or apolynucleotide encoding Tcf-4; or a vector comprising a polynucleotideencoding PIAS and/or a vector comprising a polynucleotide encodingTcf-4.
 31. The reagent kit according to claim 30, wherein the PIAS(protein inhibitor of activated STAT) is PIASy, PIAS1, PIAS3, or PIASxα.32. The reagent kit according to claim 30, wherein the PIAS (proteininhibitor of activated STAT) is PIASy.
 33. A method for inhibitingtranscriptional activity of Tcf-4, comprising desumoylating Tcf-4sumoylated by PIAS (protein inhibitor of activated STAT), wherein thedesumoylating of Tcf-4 is carried out by using Axam or an Axam mutantthat has desmoylation activity.
 34. The method for inhibitingtranscriptional activity of Tcf-4 according to claim 33, wherein theAxam mutant that has desmoylation activity is a deletion mutantcomprising the amino acid residues from the position 72 to the position588 or the amino acid residues from the position 381 to the position 588in the amino acid sequence of Axam.
 35. A method for preventing and/ortreating colon cancer, comprising desumoylating Tcf-4 sumoylated by PIAS(protein inhibitor of activated STAT), wherein the desumoylating ofTcf-4 is carried by using Axam or an Axam mutant that has desumoylationactivity.
 36. A deletion mutant of Axam comprising the amino acidresidues from the position 72 to the position 588 or the amino acidresidues from the position 381 to the position 588 in the amino acidsequence of Axam, wherein the deletion mutant has a function to inhibitthe sumoylation of Tcf-4 by PIAS (protein inhibitor of activated STAT).37. An inhibitor of transcriptional activity of Tcf-4, containing thedeletion mutant according to claim
 36. 38. A method for preventingand/or treating colon cancer, comprising administering the deletionmutant of claim 36 or a composition containing said deletion mutant.